This research will be done primarily in Spain at Universidad de Complutense Madrid in collaboration with Dr. Cesar Nombela as an extension of NIH grant R01 DE14029. The hypothesis of the parent grant and this FIRCA application is that alterations in gene expression accompany the change of Candida albicans from a free living planktonic cell to a sessile member of a biofilm. The parent grant focuses on expression profiling (microarrays) in mono- and mixed species biofilm and in organisms recovered from the oral cavity (Aims 1 and 2) and analysis of selected regulated genes through generation of deficient mutant strains (Aim 3). This FIRCA project is a proteomic project to examine protein expression and identify differences between planktonic and sessile organisms. Nombela and colleagues have established the feasibility of a proteomics approach with studies of proteins in cell wall subfractions of C. albicans yeast cells and germ tubes and have used mass spectrophotometric (MS) techniques (MALDI-TOF) for identification of cell wall proteins from Saccharomyces cerevisiae and C. albicans cytoplasm Cell wall and cytoplasmic extracts will be examined and, time permitting other subcellular fractions may be examined if indicated by expression analysis derived from the parent grant. In Aim 1, the profiles of extracts from biofilm organisms will be compared to that of yeast cells and germ tubes. This complements the first aim of the parent grant. In Aim 2, the examination will be extended to extracts from selected mutant strains. This complements the third aim of the parent grant in which the mutant strains are constructed and examined for biofilm formation and global gene expression. Proteins will be separated by 2D-PAGE and profiles compared to identify qualitative and quantitative differences in protein abundance. Proteins that differ between planktonic and biofilm organisms or wild type and mutant strain will be identified by mass spectrometric methods with priority given to differences in Aim 1 profiles.